Esther M. González

The Response of Carbon Metabolism and Antioxidant Defenses of Alfalfa Nodules to Drought Stress and to the Subsequent Recovery of Plants

Alfalfa (Medicago sativa) plants were exposed to drought to examine the involvement of carbon metabolism and oxidative stress in the decline of nitrogenase (N2ase) activity. Exposure of plants to a moderate drought (leaf water potential of −1.3 MPa) had no effect on sucrose (Suc) synthase (SS) activity, but caused inhibition of N2ase activity (−43%), accumulation of succinate (+36%) and Suc (+58%), and up-regulation of genes encoding cytosolic CuZn-superoxide dismutase (SOD), plastid FeSOD, cytosolic glutathione reductase, and bacterial MnSOD and catalases B and C. Intensification of stress (−2.1 MPa) decreased N2ase (−82%) and SS (−30%) activities and increased malate (+40%), succinate (+68%), and Suc (+435%). There was also up-regulation (mRNA) of cytosolic ascorbate peroxidase and down-regulation (mRNA) of SS, homoglutathione synthetase, and bacterial catalase A. Drought stress did not affect nifH mRNA level or leghemoglobin expression, but decreased MoFe- and Fe-proteins. Rewatering of plants led to a partial recovery of the activity (75%) and proteins (>64%) of N2ase, a complete recovery of Suc, and a decrease of malate (−48%) relative to control. The increase in O2 diffusion resistance, the decrease in N2ase-linked respiration and N2ase proteins, the accumulation of respiratory substrates and oxidized lipids and proteins, and the up-regulation of antioxidant genes reveal that bacteroids have their respiratory activity impaired and that oxidative stress occurs in nodules under drought conditions prior to any detectable effect on SS or leghemoglobin. We conclude that a limitation in metabolic capacity of bacteroids and oxidative damage of cellular components are contributing factors to the inhibition of N2ase activity in alfalfa nodules.

Medicago truncatula Root Nodule Proteome Analysis Reveals Differential Plant and Bacteroid Responses to Drought Stress

Drought is one of the environmental factors most affecting crop production. Under drought, symbiotic nitrogen fixation is one of the physiological processes to first show stress responses in nodulated legumes. This inhibition process involves a number of factors whose interactions are not yet understood. This work aims to further understand changes occurring in nodules under drought stress from a proteomic perspective. Drought was imposed on Medicago truncatula ‘Jemalong A17’ plants grown in symbiosis with Sinorhizobium meliloti strain 2011. Changes at the protein level were analyzed using a nongel approach based on liquid chromatography coupled to tandem mass spectrometry. Due to the complexity of nodule tissue, the separation of plant and bacteroid fractions in M. truncatula root nodules was first checked with the aim of minimizing cross contamination between the fractions. Second, the protein plant fraction of M. truncatula nodules was profiled, leading to the identification of 377 plant proteins, the largest description of the plant nodule proteome so far. Third, both symbiotic partners were independently analyzed for quantitative differences at the protein level during drought stress. Multivariate data mining allowed for the classification of proteins sets that were involved in drought stress responses. The isolation of the nodule plant and bacteroid protein fractions enabled the independent analysis of the response of both counterparts, gaining further understanding of how each symbiotic member is distinctly affected at the protein level under a water-deficit situation.

Nitrogen Fixation Control under Drought Stress. Localized or Systemic

Legume-Rhizobium nitrogen fixation is dramatically affected under drought and other environmental constraints. However, it has yet to be established as to whether such regulation of nitrogen fixation is only exerted at the whole-plant level (e.g. by a systemic nitrogen feedback mechanism) or can also occur at a local nodule level. To address this question, nodulated pea (Pisum sativum) plants were grown in a split-root system, which allowed for half of the root system to be irrigated at field capacity, while the other half was water deprived, thus provoking changes in the nodule water potential. Nitrogen fixation only declined in the water-deprived, half-root system and this result was correlated with modifications in the activities of key nodules enzymes such as sucrose synthase and isocitrate dehydrogenase and in nodular malate content. Furthermore, the decline in nodule water potential resulted in a cell redox imbalance. The results also indicate that systemic nitrogen feedback signaling was not operating in these water-stressed plants, since nitrogen fixation activity was maintained at control values in the watered half of the split-root plants. Thus, the use of a partially droughted split-root system provides evidence that nitrogen fixation activity under drought stress is mainly controlled at the local level rather than by a systemic nitrogen signal.

Reduced Carbon Availability to Bacteroids and Elevated Ureides in Nodules, But Not in Shoots, Are Involved in the Nitrogen Fixation Response to Early Drought in Soybean

Nitrogen fixation (NF) in soybean (Glycine max L. Merr.) is highly sensitive to soil drying. This sensitivity has been related to an accumulation of nitrogen compounds, either in shoots or in nodules, and a nodular carbon flux shortage under drought. To assess the relative importance of carbon and nitrogen status on NF regulation, the responses to the early stages of drought were monitored with two soybean cultivars with known contrasting tolerance to drought. In the sensitive cultivar (‘Biloxi’), NF inhibition occurred earlier and was more dramatic than in the tolerant cultivar (‘Jackson’). The carbon flux to bacteroids was also more affected in ‘Biloxi’ than in ‘Jackson’, due to an earlier inhibition of sucrose synthase activity and a larger decrease of malate concentration in the former. Drought provoked ureide accumulation in nodules of both cultivars, but this accumulation was higher and occurred earlier in ‘Biloxi’. However, at this early stage of drought, there was no accumulation of ureides in the leaves of either cultivar. These results indicate that a combination of both reduced carbon flux and nitrogen accumulation in nodules, but not in shoots, is involved in the inhibition of NF in soybean under early drought.

Carbon metabolism and bacteroid functioning are involved in the regulation of nitrogen fixation in Medicago truncatula under drought and recovery.

Regulation of symbiotic nitrogen fixation (SNF) during drought stress is complex and not yet fully understood. In the present work, the involvement of nodule C and N metabolism in the regulation of SNF in Medicago truncatula under drought and a subsequent rewatering treatment was analyzed using a combination of metabolomic and proteomic approaches. Drought induced a reduction of SNF rates and major changes in the metabolic profile of nodules, mostly an accumulation of amino acids (Pro, His, and Trp) and carbohydrates (sucrose, galactinol, raffinose, and trehalose). This accumulation was coincidental with a decline in the levels of bacteroid proteins involved in SNF and C metabolism, along with a partial reduction of the levels of plant sucrose synthase 1 (SuSy1). In contrast, the variations in enzymes related to N assimilation were found not to correlate with the reduction in SNF, suggesting that these enzymes do not have a role in the regulation of SNF. Unlike the situation in other legumes such as pea and soybean, the drought-induced inhibition of SNF in M. truncatula appears to be caused by impairment of bacteroid metabolism and N(2)-fixing capacity rather than a limitation of respiratory substrate.

Abiotic Stress Responses in Legumes: Strategies Used to Cope with Environmental Challenges

Legumes are well recognized for their nutritional and health benefits as well as for their impact in the sustainability of agricultural systems. The threatening scenario imposed by climate change highlights the need for concerted research approaches in order to develop crops that are able to cope with environmental stresses, while increasing yield and quality. During the last decade, some physiological components and molecular players underlying abiotic stress responses of a broad range of legume species have been elucidated. Plant physiology approaches provided general outlines of plant responses, identifying stress tolerance-related traits or elite cultivars. A thorough identification of candidate genes and quantitative trait loci (QTLs) associated with these traits followed. Model legumes like Medicago truncatula, Lotus japonicus, and more recently, Glycine max provided valuable translational approaches for dissecting legume responses to abiotic stresses. The challenge now focuses on the translation of the information gained in model systems in controlled environments to crops grown under field conditions. In this review, we provide a general overview of the recent achievements on the study of abiotic stress responses in a broad range of model, grain and forage legumes species, highlighting the different approaches used. Major accomplishments, as well as limitations or drawbacks are discussed across the different sections. Some perspectives regarding new approaches for screening, breeding or engineering legumes with desirable abiotic stress resistance traits are anticipated. These advances will support the development of legumes better adapted to environmental constraints, tackling current demands on modern agriculture and food production presently exacerbated by global climate changes.

Local inhibition of nitrogen fixation and nodule metabolism in drought-stressed soybean

Drought stress is a major factor limiting symbiotic nitrogen fixation (NF) in soybean crop production. However, the regulatory mechanisms involved in this inhibition are still controversial. Soybean plants were symbiotically grown in a split-root system (SRS), which allowed for half of the root system to be irrigated at field capacity while the other half remained water deprived. NF declined in the water-deprived root system while nitrogenase activity was maintained at control values in the well-watered half. Concomitantly, amino acids and ureides accumulated in the water-deprived belowground organs regardless of transpiration rates. Ureide accumulation was found to be related to the decline in their degradation activities rather than increased biosynthesis. Finally, proteomic analysis suggests that plant carbon metabolism, protein synthesis, amino acid metabolism, and cell growth are among the processes most altered in soybean nodules under drought stress. Results presented here support the hypothesis of a local regulation of NF taking place in soybean and downplay the role of ureides in the inhibition of NF.

Comparative transcriptomic analysis of salt adaptation in roots of contrasting Medicago truncatula genotypes.

Evolutionary diversity can be driven by the interaction of plants with different environments. Molecular bases involved in ecological adaptations to abiotic constraints can be explored using genomic tools. Legumes are major crops worldwide and soil salinity is a main stress affecting yield in these plants. We analyzed in the Medicago truncatula legume the root transcriptome of two genotypes having contrasting responses to salt stress: TN1.11, sampled in a salty Tunisian soil, and the reference Jemalong A17 genotype. TN1.11 plants show increased root growth under salt stress as well as a differential accumulation of sodium ions when compared to A17. Transcriptomic analysis revealed specific gene clusters preferentially regulated by salt in root apices of TN1.11, notably those related to the auxin pathway and to changes in histone variant isoforms. Many genes encoding transcription factors (TFs) were also differentially regulated between the two genotypes in response to salt. Among those selected for functional studies, overexpression in roots of the A17 genotype of the bHLH-type TF most differentially regulated between genotypes improved significantly root growth under salt stress. Despite the global complexity of the differential transcriptional responses, we propose that an increase in this bHLH TF expression may be linked to the adaptation of M. truncatula to saline soil environments.

Physiological consequences of continuous, sublethal imazethapyr supply to pea plants

Summary Imazethapyr (IM) is a herbicide that inhibits the branched-chain amino acid (BCAA) biosynthesis through the specific inhibition of acetolactate synthase activity. This herbicide acts very slowly and several weeks are required for complete plant death. From the BCAA biosynthesis inhibition to the growth inhibition and plant death, the processes involved are not fully understood. Starvation for BCAAs and/or starvation for carbohydrates in sinks. have been proposed as part of the death mechanisms. In this study, a permanent acetolactate synthase inhibition is used in order to (1) determine whether the growth inhibition effects can be attributed to a reduction in BCAA content and/or to starvation of carbohydrates; and (2) to analyse the physiological changes induced. Sublethal doses of IM were continuously supplied in the nutrient solution of nodulated pea plants. These conditions led to a significant decline in plant growth. The herbicide also caused a decline in nodule initiation, but had little effect on nodule development. However, plants were not nitrogen-limited and net photosynthesis was only slightly affected at the higher herbicide concentration. Total soluble sugars and starch were accumulated in both leaves and roots following herbicide supply. These results were also found in non-nodulated, nitrate-fed plants. In relation with a likely BCAA starvation, a significant increase was observed in the free amino acid pool, with a marked imbalance among different amino acids, although among BCAAs, only valine pool declined as a consequence of IM supply. It is concluded that acetolactate synthase inhibition by continuous, sublethal IM supply does not induce carbohydrate or a specific BCAA starvation in pea plants.

Source of nitrogen nutrition (nitrogen fixation or nitrate assimilation) is a major factor involved in pea response to moderate water stress.

Summary The effect of the source of nitrogen nutrition (nitrogen fixation or nitrate assimilation) on the response of pea plants to a gradual and moderate water stress was studied. Growth declined under water deficit, but nodulated plants were less sensitive to drought than nitrate-fed plants. Stomatal conductance and internal CO 2 concentration also decreased, but both were higher in nitrogen-fixing plants throughout the drought period, leading to better maintenance of carbon assimilation rates under water deficit. Glycolate oxidase, a key enzyme in the photorespiratory cycle, declined by 50% in nitrogen-fixing plants under water deficit, although it was not affected in nitrate-fed plants. Nitrogen assimilation declined during the drought period and was independent of nitrogen source. Free amino acid content declined in leaves of plants grown under both nutrition regimes, reflecting the decrease in nitrogen assimilation. Water stress led to carbohydrate accumulation in pea plants grown with either nitrogen source, but it was higher in nitrogen-fixing plants. Roots showed the greatest carbohydrate and amino acid accumulation in both nutritions regimes, with significantly greater increases in free amino acids in nitrate-fed plants. It is concluded that the nitrogen source is a major factor affecting pea responses to water stress, although the difference in sensitivity seems to be related not to the nitrogen assimilation process but to complex interactions with photorespiratory flux and stomatal conductance.